Clue to previous blogpost "Enzymes...have you wondered"

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Lecture test 2 Q2d: How pH changes affect enzymatic reaction

I have finished marking the lecture tests and I wish to highlight one question that was very poorly attempted in the test here. Although most of you are already aware that a change in pH from the optimum (more than or less than optimum) decreases enzyme activity, alot of you were unable to explain the mechanism behind this effect. Actually, the mechanism is already spelt out very clearly in your lecture notes so please go over it again to make sure you are clear about what I am saying here.

I also want to clarify some terms here as some of you seem to be confused over these terms. The enzyme is a globular protein that has undergone FOLDING to achieve its respective secondary/tertiary/quaternary (if more than one polypeptide chain) structures. Hence it has a specific CONFORMATION/configuration/3D structure as a result of the folding. And it is due to this conformation that the enzyme has a specific SHAPE in its ACTIVE SITE, which is complementary to the shape of its substrate. To summarise, "conformation" is not the same as "shape" so please don't use them interchangeably. The enzyme has a specific "conformation" while the active site has a specific "shape". Hope that this is clear.

Now let's move on to the mechanism. As I have already mentioned in class,  a change in pH changes the concentration of H+ and OH- ions, which causes the neutralisation of polar/charged R groups in the enzyme.

But what you should also know is that this effect can occur at the follwoing 2 areas of the enzyme. 

(i) At the active site -

At the active site, the catalytic and contact residues have charged R groups.

Hence, NEUTRALISATION of these CHARGED R groups disrupts the formation of INTERmolecular IONIC AND HYDROGEN bonds between the active site on the ENZYME and the SUBSTRATE. 

(ii) At other regions of the enzyme -

The bulk of the globular structure of the enzyme contain polar R groups (that form hydrogen bonds) and charged R groups (that form ionic bonds), which stabilise/maintain the secondary, tertiary and/or quaternary structures of the enzyme.

Hence, NEUTRALISATION of these POLAR/CHARGED R groups disrupts the INTRAmolecular IONIC AND HYDROGEN bonds maintaining the SECONDARY, TERTIARY and/or QUATERNARY structure of the enzyme. This causes the enzyme to unfold and lose its specific conformation (ie. denaturation) of the enzyme, hence causing the active site to lose its specific shape.

With both disruptions, binding of the substrate to the active site of the enzyme to form enzyme-substrate complexes, and hence products are prevented. And that is why the enzymatic activity decreases!

Enzymes tutorial Q4b - Although Km is not in your syllabus, you still have to draw the curves accurately!

Enzymes tutorial Q2ciii

Q) Suggest why the lysosomal membrane remains intact? [3]

Possible Answers:

[1st mark] 
- Lysosomal enzymes hydrolyse polysaccharides, lipids, proteins, and nucleic acids.

[2nd mark] 
- These differ from the constituents of the lysosomal membrane which comprise of phospholipids, membrane-bound proteins, glycolipids, and glycoproteins. [1]
OR 
- The inner surface of the lysosomal membrane is coated with an extensive glycocalyx (refer to pictures below).
OR
- The integral and inner peripheral membrane proteins and membrane phospholipids on the inner surface of the lysosomal membrane are highly glycosylated.
OR 
- There are large numbers of glycoproteins and glycolipids on the inner surface of the lysosomal membrane.

[3rd mark]
- As the constituents of the lysosomal membrane are not complementary to the active sites of the lysosomal enzymes, they cannot fit into the active sites and thus, remain unhydrolysed. [1]
OR
- The glycosylation on the inner lysosomal membrane prevents the lysosomal enzymes from accessing the membrane phospholipids and proteins.

Enzymes - Have you wondered...

1) How enzymes from thermophilic bacteria is able to remain stable/retain catalytic activity at high temperatures?

2) Why lysosomal enzymes can function at a pH of 5.5 but not at pH of 6.8, even though the numbers are quite close?

Any takers?

Enzymes worksheet B (temperature) - Q1b

Please be specific in your answer/ note the context of the question (i.e. NAME the enzyme and substrate if the question stem mentions them)

- D: Initially at very low temperature, the enzyme activity (rate of enzymatic reaction) is very low.
- E: This is due to the inactivation of the enzymes, and the low kinetic energies of both the substrate and enzyme molecules, leading to a low frequency of effective collisions between them, and hence reduced rate of formation of enzyme-substrate complexes and products. 

- D: As temperature increases to optimum, the enzyme activity (rate) doubles for every 10C increase in temperature (Q10), and eventually reaches a peak/maximum rate at the optimum temperature (65C). 
- E: Increased temperature increases the kinetic energies of both enzyme and substrate molecules which in turn, increases the frequency of effective collisions between them. As a result, more enzyme-substrate complexes and hence products are formed.

- D: As temperature increases to beyond optimum, the enzyme activity (rate) decreases.
- E: This is due to the excess heat increases the vibrations of atoms within enzyme and disrupts the intramolecular hydrogen bonds, ionic bonds, and hydrophobic/hydrophilic interactions stabilising the secondary and tertiary structures of the enzymes, causing it to unfold and loses its specific 3D configuration/conformation (enzyme denatures). Hence its active site loses its specific shape, causing the substrate to be unable to bind to it. Hence, less enzyme-substrate complexes and products are formed.

P.S. If you have any queries with regards to the 3 MCQs in this enzymes wksheet B, you can ask me under comments here, or during the next tutorial. Note that we will be working on the main enzymes tutorial next week so please complete it over the wkend (treat it as lecture test revision). I aim to finish Enzymes before I leave! Hope you have enjoyed and benefited from my lessons. Don't miss me.. HAHA! See you next week! :)